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Schwann cells from neonatal rat sciatic nerve can be maintained and grown in culture in the absence of neurons. We are interested in substantially expanding such cultures for use in the study of Schwann cells, their growth responses, and their interactions with neurons. However, it was important to determine if expanded cell populations retained their distinguishing biological properties and their ability to differentiate when recombined with neurons. Therefore, we have compared the functional properties of extensively expanded populations of sciatic nerve Schwann cells to those of embryonic dorsal root ganglion (DRG) Schwann cells that had been briefly expanded in vitro in the continuous presence of ganglion neurons. Sciatic nerve Schwann cells were cultured and purified according to the methods of Brockes et al. (1979). A combination of crude glial growth factor and forskolin was found to act synergistically in providing maximal stimulation of Schwann cell proliferation. Sciatic nerve Schwann cells that were continuously expanded for at least 2 months were compared to Schwann cells derived from fetal dorsal root ganglia. The results indicate that the complement of secreted proteins from both cell populations, either in isolation or recombined with neurons, was essentially identical; both cell populations expressed the cell-surface antigens laminin and Ran 1 (217C antibody); after seeding onto DRG neurons, both cell populations associated with neuronal processes with the same time course; and under identical nutrient conditions, both cell populations were observed to exhibit a comparable capacity for myelination of DRG axons in vitro. Thus, methods used to establish primary cultures of rat sciatic nerve Schwann cells and to expand secondary cultures in vitro in the absence of neurons preserve basic Schwann cell functions.

Myristoyl CoA:protein N-myristoyltransferase (NMT) catalyzes the addition of myristic acid to the amino-terminal glycine residues of a number of eukaryotic proteins. Recently, we developed a cell-free system for analyzing NMT activity and have begun to characterize the substrate specificity of this enzyme by using a series of synthetic peptides. We have now purified NMT from Saccharomyces cerevisiae to apparent homogeneity. The native enzyme is a 55-kDa protein, exhibits no requirement for divalent cation, and appears to contain a histidine residue critical for enzyme activity. A total of 42 synthetic peptides have been used to define structure/activity relationships in NMT substrates. An amino-terminal glycine is required for acylation; substitution with glycine analogues produces peptides that are inactive as substrates or inhibitors of NMT. A broad spectrum of amino acids is permitted at positions 3 and 4, while strict amino acid requirements are exhibited at position 5. Replacement of Ala5 in the peptide Gly-Asn-Ala-Ala-Ala-Ala-Arg-Arg with Asp ablates the peptide's myristoyl-accepting activity. A serine at this position results in a decrease by a factor of approximately equal to 500 in the apparent Km in the context of three different sequences. Penta- and hexa-peptides are substrates, but with decreased affinity. These studies establish that structural information important for NMT-ligand interaction exists beyond the first two amino acids in peptide substrates and that the side chains of residue 5 play a critical role in the binding of substrates to this enzyme.

In the preceding paper (Salzer et al ., 1980, J. Cell Biol. 84:753-766), evidence was presented that a neurite membrane fraction could be used to stimulate Schwann cell proliferation in culture . In this study, we present evidence that the mitogenic signal by which intact neurites or neurite membranes stimulate Schwann cell proliferation is located at the neurite surface . This conclusion is based on the following observations : (a) stimulation of Schwann cell proliferation by neurons requires direct contact between neurites and Schwann cells, separation of the two cells by a permeable collagen diaphragm 6 ,um thick prevents Schwann cell proliferation ; (b) treatment of intact neurites with trypsin before preparation of neurite membranes abolishes the ability of these membranes to be mitogenic for Schwann cells; and (c) the mitogenic activity of neurite homogenates is exclusively localized in the particulate rather than the soluble fraction of the homogenate .The mitogenic component on the neurite surface is heat labile, and is inactivated by aldehyde fixation . Preliminary data suggest that the mitogenic effect of neurite on Schwann cells is not mediated by 3',5'-cyclic AMP .KEY WORDS Schwann cells " dorsal root ganglia -mitogenic response cell interactions " membrane localizationThe development of pure populations of neurons or non-neuronal cells has recently provided unique insights into the normal cellular interactions of the peripheral nervous system (3) . One such system described by Wood (38) allows the preparation of neurons and Schwann cells in isolation and led to the discovery that sensory neurites provide a mitogenic stimulus for Schwann cell division (39) . A similar conclusion regarding growth control of non-neuronal cells from the chick sympathetic nervous system was reached by McCarthy and Partlow (20) . J . CELL BIOLOGY

When prepared by methods utilized in our laboratory, pure populations of Schwann cells in culture do not divide, but, after recombination with peripheral sensory neurons or their processes, proliferate rapidly (Wood and Bunge, 1975, Nature (Lond) 256:661-664). In this paper, we demonstrate that a membrane fraction prepared from sensory ganglion neurites is also mitogenic for Schwann KEY WORDS Schwann cell proliferation mitogenic signal . nerve tissue culture membranes " autoradiography In the first paper of this series we have reviewed the evidence that proliferation of Schwann cells during development is dependent on a mitogenic signal provided by the growing axon (19) . Evidence is also presented that this stimulus for Schwann cell proliferation is distinct from that observed when Schwann cells proliferate during Wallerian degeneration initiated by axotomy . Modifying described culture techniques (27), we J . CELL BIOLOGY